Klemets R, Lundberg B
Lipids. 1984 Sep;19(9):692-8. doi: 10.1007/BF02534529.
Ion-exchange chromatography and preparative isoelectric focusing (PIEF) were compared to produce a stable rat liver lysosomal cholesteryl ester hydrolase of high specific activity. The PIEF purification method proved to be more rapid and easier to perform. PIEF purification involved the following steps: i) osmotic shock of the lysosome fraction, ii) (NH4)2 SO4 precipitation (10-70%, w/v), iii) Sepharose CL-6B gel filtration, and iv) PIEF. The enzyme was purified 60-120-fold with a yield of 2-4%. The activity of the purified enzyme was best restored by stabilizing with a 0.5% (w/v) albumin solution. The purified enzyme produced one major band on SDS-polyacrylamide gel electrophoresis having a MW of 58,500 daltons. Gel filtration showed a MW of 58,000 daltons. The optimum pH of the enzyme was 4.5, and the isoelectric point was 6.0-6.2. The specific activity of hydrolysis of cholesteryl oleate and triolein increased by similar rates during purification.
比较了离子交换色谱法和制备性等电聚焦（PIEF）法，以制备具有高比活性的稳定大鼠肝脏溶酶体胆固醇酯水解酶。结果证明PIEF纯化方法更快且更易于操作。PIEF纯化包括以下步骤：i）对溶酶体部分进行渗透压休克；ii）硫酸铵沉淀（10 - 70%，w/v）；iii）琼脂糖CL - 6B凝胶过滤；iv）PIEF。该酶纯化了60 - 120倍，产率为2 - 4%。用0.5%（w/v）白蛋白溶液稳定后，纯化酶的活性得到最佳恢复。纯化后的酶在SDS - 聚丙烯酰胺凝胶电泳上产生一条主要条带，分子量为58,500道尔顿。凝胶过滤显示分子量为58,000道尔顿。该酶的最适pH为4.5，等电点为6.0 - 6.2。在纯化过程中，胆固醇油酸酯和三油精的水解比活性以相似的速率增加。
